K. Rool himself from DK64. The ominous music is a bonus, but the voice acting in the intro is a bit of Nightmare Retardant given its similarities to Dr. Claw from Inspector Gadget.

Dogadon in Fungi Forest: after dealing a certain amount of damage to him, he starts making the platform sink, as the music gets faster and faster as the platform gets lower.

It's entirely possible for the platform to fully sink into the lava. If you take too long with the fight, it will end with him laughing and flying away, as Chunky is left to burn.

The wind-up Kremlings from Frantic Factory are Nightmare Fuel incarnate. Everything about them is unsettling: glowing red eyes, circular walking pattern when not seeing you, squeaky noises when seeing you.

Creepy Castle has rotting hands as platforms. Inside the actual castle, there's a torture chamber after a dark maze and a very realistic skull-shaped portal leading to a red maze made completely out of bones.

Creepy Castle had an unseen individual moaning constantly.

Angry Aztec has an eerie temple with ominous music that just makes you feel uncomfortable, and once you grab the banana at the end, a voice suddenly shouts "GET OUT!" and aims at you with some sort of gun. You then have 25 seconds to get out of there before being shot down. Said "GET OUT!" comes from an assassin who tries to kill you once you get the bananas. In certain Crystal Caves challenges and in the greenhouse in Creepy Castle, he returns if you fail a challenge and gives you only 10 seconds to escape this time.

One Chunky Kong challenge in Crystal Caves has you avoiding lights. Hit a light, and you get killed instantly after one second!

The Crystal Caves seem like a relatively calm place at first, it's very ambient and you're just walking around gazing at the serenity of the area until a scary noise accompanies stalactites falling everywhere.

Good news: it's possible for Tiny Kong to locate the source of the falling stalactites. The bad news: it's a gigantic Kosha, which is about ten times her size! He still goes down normally, but YEESH!

The fact that the entire Hideout Helm level was timed, considering that if time runs out, it's an automatic game over and all of DK Isles gets destroyed.

The Evil Laughter that occurs throughout the game (mostly when you use a barrel or animal ability where it isn't allowed) is heard within every 10 minute interval in the level. By the time you reach the last 10 minutes, the laughing becomes even more frequent and starts occurring every minute. When you reach the last 10 seconds, the game counts down directly on the screen...

The Game Over screen (thankfully skippable, though) has the evil laughter K. Rool showing the Blast-O-Matic about to blow DK Isles up, which was pretty intense for some back when they were children, joining the ranks of Banjo-Kazooie in the "save and quit is not an option" territory.

The Golden Banana where you have to swim into the giant mechanical fish. Not helped by the fact that a late-game glitch renders the banana almost impossible to obtain after getting the Sniper Scope if you don't know how to circumvent it.

Anytime you asked Wrinkly - Cranky's dead wife - for hints, she would always enter with her wailing.

Wrinkly is heavily implied to have died between Donkey Kong Country 3 and DK64.

The constant crying of the Banana Fairy is unnerving as well.

The Evil Laughter that occurs throughout the game is nerve-wracking, especially when you least expect it, like when using a barrel ability or an animal pal ability where it's not allowed.

The Green Klaptraps in Angry Aztec wear snapping dentures with tongues in them that give chase after you should you kill one.

If you didn't know any better, you'd think that you knocked their teeth and tongue out directly, with these chasing you for one last attempt at revenge from beyond the grave!

Fungi Forest at night is pretty scary to those unfamiliar with the level layout.

Creepy Castle's mine cart section. It's bad enough with the loud chase music and random sirens going off constantly, but how about the tombstones that fly out the ground (with accompanying insane laughter) as you approach or the giant skeleton demon thing shooting explosive fireballs at poor DK?

The tunnels in Jungle Japes and Gloomy Galleon have whispers echo throughout.

The secret cavern reached in Jungle Japes by pounding a large X as Chunky has a narrow path above a Bottomless Pit lined with massive torches and a giant skull at the end. The music certainly doesn't help matters.

Said creepy music is reused in a beehive within the same level, and the ghost ship in Gloomy Galleon.

As Donkey Kong, you have to complete a few Barrel Cannon challenges that are set in the sky, while a windy and lonely rendition of the aforementioned cave music plays. These challenges tend to invoke feelings of acrophobia.

Zingers are back, and you can shoot them down. Not scary? Well then, try aiming at one at first-person close enough for it to bear down on you. The only thing scarier than regular Zingers are the mechanical Zingers in Frantic Factory. Oh jeez...

And later they start dropping grenades at you, making the task of getting past them from below incredibly unnerving as you hear that falling sound followed by a loud explosion that might get you by surprise.

The game gives you Super Not-Drowning Skills, as per Donkey Kong Country tradition. However, Rareware seems to have used this as an excuse to make some very deep hydrophobe-unfriendly water areas, such as those in Gloomy Galleon.

The sunken ship rooms are probably the worst thanks to the bad lighting, killer starfish and exploding (and creepy looking) pufferfish (both of those enemies can't be fought due to the Kongs lacking underwater combat moves). Some of those rooms actually feature jail cells and skeletons of prisoners chained to walls, because it can always get worse.

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